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Anjali Singh Author
Anjali Singh is a freelance writer. Following her passion for science and research she did her Master’s in Plant Biology and Biotechnology from the University of Hyderabad, India. She has a strong research background in Plant Sciences with expertise in Molecular techniques, Tissue culture, and Biochemical Assays. In her free time outside work, she likes to read fictional books, sketch, or write poems. In the future, she aspires to pursue a doctorate in Cancer Biology while continuing her excellence as a scientific writer.
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Anjali Singh Author
Anjali Singh is a freelance writer. Following her passion for science and research she did her Master’s in Plant Biology and Biotechnology from the University of Hyderabad, India. She has a strong research background in Plant Sciences with expertise in Molecular techniques, Tissue culture, and Biochemical Assays. In her free time outside work, she likes to read fictional books, sketch, or write poems. In the future, she aspires to pursue a doctorate in Cancer Biology while continuing her excellence as a scientific writer.
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  • Nucleic Acid Quantification Protocols
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Introduction

Fungi is a group of eukaryotic organisms that include a myriad of single and multicellular organisms that are parasitic or saprophytic in nature. Over 1.5 million species of fungus have been discovered and they are found in a range of habitats. Some characteristic feature of fungus include:

  • Ubiquitous
  • Eukaryotic (uninucleate or multinucleate)
  • Thallus body (Composed of tube-like structures called hyphae) – except yeast (single-celled)
  • Lack chlorophyll
  • Spore-producing organisms
  • Mode of reproduction: Sexual or asexual

Ubiquitous is an accurate description for them because of the diversity in their habitats, including extreme environments such as high salt concentration or deep sediments. Although fungi are mostly known in vexing ways, since they cause diseases in plants (such as rust and smut) and in human beings (infections), they are also an essential element of the ecosystem and play numerous important roles that include:

  • Biological insecticide: Trichoderma species and Beauveria bassiana.
  • Industrial processes: It includes fermentation of beer and wine and in the making of bread (for example, Saccharomyces cerevisiae-yeast).
  • Use as food: Such as mushroom and some morels and truffles.
  • Medicine: Many secondary metabolites of fungi are used for medicinal purposes. A few examples of drugs isolated from fungi are cyclosporin, cephalosporin, and psilocybin.
  • Research: To understand the eukaryotic genome. For example, Saccharomyces cerevisiae, Saccharomyces pombe, Neurospora crassa, and Aspergillus nidulans.

Because of the functional diverse roles of fungi in the ecosystem, plants (symbiosis), and pharmaceuticals (to make antibiotics), they have become one of the favorite areas of study for researchers.

In this article, we will study the laboratory culturing methods of a few fungi and cytogenetic techniques to demonstrate fungal chromosomes.

Cytogenetics of fungus

A number of fungi are used as model organisms in the labs to understand various fundamental processes and genetics of eukaryotic organisms, such as to study gene regulation, chromatin structure, and cell cycle regulation. Different model organisms serve different research purposes. For example:

  1. Yeast (Saccharomyces cerevisiae & Saccharomyces pombe): to study the meiotic and epigenetic machinery[7].
  2. Neurospora crassa: to study the epigenetic phenomenon[7].
  3. Sordaria fimicola: for meiosis and genetic diversity
  4. Aspergillus nidulans: to study gene regulation

But the question arises “what makes them model organisms?”

There are some characteristic features of a few fungi that make them the “model” for laboratories, including[9]:

  • Less expensive to maintain and culture in lab condition (in comparison to other eukaryotic organisms).
  • Short generation time.
  • Very large numbers of progeny from each cross.
  • Easily available strains.
  • Small genome size.
  • Fewer chromosome numbers.
  • Entire sequenced genome.
  • Haploid life cycle.

Before we move to the fungal cytogenetic methods, it is critical to understand the culturing methods of fungi in laboratories.

Growth and maintenance of fungi under laboratory conditions

The following includes the medium required to culture different fungi under laboratory conditions. It should be noted that as different fungi thrive in variant habitats, they may require different media components to be cultured in labs.

1.     Saccharomyces cerevisiae (yeast) culturing methods

There are four types of culture media to grow yeast in labs. They are YPD or YEPD, Synthetic culture media, minimal media, and sporulation media. The choice of media depends on the type of experiment to be done. The method below details how to grow yeast in synthetic culture media[8].

Synthetic culture media

Material

S, cerevisiae, Bioreactor, 10 g KH2PO4, 4 g (NH4)2SO4, 0.8 g MgSO4, 2 g yeast extract, 10 g glucose, physiological serum, 2 L deionized water, and HCl (1M) (to adjust the pH)[8].

Bioreactor

It is a device that is used to support a biologically active environment for the growth of an organism under laboratory conditions.

It is composed of three parts: controller, gas mixer, and gas analyzer. It contains a double jacket vessel, around which water circulates between the two jackets. The temperature of the water circulating around the vessel should be 10-15 ℃ lower than the synthetic media to be used for yeast growth[8].

Preparation of synthetic media

Mix exact concentration of the given chemicals in a flask: 10 g KH2PO4, 4 g (NH4)2SO4, 0.8 g MgSO4, 2 g yeast extract, and 10 g glucose in 2 L deionized water[8].

Preparation of suspension of yeast cell

Add yeast cell (108 CFU/ml) in 3 ml physiologic serum.

Procedure

  1. Fill the bioreactor with 2 L of synthetic culture media (when the bioreactor vessel is of 3L).
  2. Add the 3 ml yeast cell suspension in the bioreactor vessel.
  3. Set the temperature at 30 ℃ and stir the medium at the rate of 200 rpm.
  4. Maintain the culture medium at pH 4 and 5 % dissolved oxygen (DO)[8].

2.     Neurospora crassa culturing methods

Two types of medium are used; Vogel’s medium N salts for the growth of Neurospora and the Synthetic cross-medium for the mating tests. Below is the recipe for Vogel’s medium N[5].

Vogel’s medium N

Material (for 1L of 50x salts)

Component[5]     Trace Elements Solution[5]  
water 750 ml   water 95 ml
Na3C6H5O7. 2H2O (trisodium citrate) 130 g   citric acid 5 g
KNO3 (potassium nitrate) 126 g   ZnSO4.7H2O (zinc sulfate) 5 g
(NH4 )H2PO4 (ammonium phosphate) 144 g   Fe(NH4 )2(SO4 )26H2O (ferrous ammonium sulfate)  1 g
KH2PO4 (potassium phosphate monobasic) 80 g   CuSO4.5H2O 250 mg
MgSO4.7H2O 10 g   MnSO4.H2O 50 mg
CaCl2. 2H2O 5 g   H3BO3 50 mg
Trace elements solution* 5 ml   Na2MoO4.2H2O (sodium molybdate) 50 mg
Biotin solution 2.5 ml   Chloroform (as preservative) A few ml

Biotin stock solution

  • Dissolve 5 mg biotin in 50 ml water or 50% ethanol. Store it at -20 °C.

Trace element solution

  • In 95 ml distilled water, add the components successively in the given order in the “trace element solution” table with continuous stirring, at room temperature.

Procedure

  1. Add the given chemicals successively in 750 ml water in the same order (as given in the table) with continuous stirring. Use the Erlenmeyer flask to prepare the solution.
  2. Heat the solution moderately to dissolve citrate and phosphate completely.
  3. Dissolve the calcium chloride separately in 20 ml water and add it slowly in the main solution.
  4. Add 5 ml chloroform as preservation and store the solution at room temperature[5].

Note:

  • The pH of the medium is 5.8. So, no need to adjust the pH of the solution.
  • Before adding the next component, it is to be assured that the first chemical is completely dissolved.
  • Autoclave the medium after adding sucrose (1-1.5%)(if adding)[5].

3.     Aspergillus nidulans culturing methods

Minimal medium(MM)

Procedure

  • Prepare 3 stock solution  Salt mix “lacking MgSO4 ” (20x) Stock, Trace element solution stock and MgSO4 Solution (200x) Stock[3].
    • Salt mix “lacking MgSO4 ” (20x): use 50 ml for 1 liter MM
      • In 800 ml of distilled water, dissolve the following salts[3] in the listed order (for 1L solution)
Components gram/L
NaNO3 (Sodium nitrate) 120.0g
KCl (Potassium chloride) 10.4g
KH2PO4 (Potassium phosphate, monobasic) 16.3g
K2HPO4 (Potassium phosphate, dibasic) 20.9g
      • Make up the volume of the final solution to 1 L by adding distilled water.
      • Autoclave the solution and store it at room temperature.
    • MgSO4 Solution (200x) Stock[3]: use 5 ml for 1 liter MM
      • Dissolve the MgSO4.7H2O (Magnesium sulfate) 10.4 g in 80 ml of distilled water for a 100 ml solution.
      • Make up the volume of the solution to 100 ml.
      • Autoclave the solution and store it at 4-8°C after opening.
    • Trace element solution stock (200ml, 1000x)[3]: use 1 ml for 1 litre MM.
      • Two solutions are prepared, namely “a” and “b”.
        1. In 80 ml distilled water, dissolve the listed salts in the given order:
Components grams
FeSO4.7H2O (Ferrous sulfate) 1.0g
EDTA 10.0g

A golden yellow solution is observed at pH 5.5.

        1. In another flask, add the following salts[3] in the given order in 80 ml of distilled water:
Components grams
ZnSO4 ·7H2O (Zinc sulfate) 4.4g
H3BO3 (Boric acid) 2.2g
MnCl2.4H2O (Manganous chloride) 1.0g
CoCl2.6H2O (Cobaltous chloride) 0.32g
CuSO4.5H2O (Cupric sulfate) 0.32g
(NH4 )6Mo7O24·4H2O (Ammonium molybdate) 0.22g
    • Mix both the “a” and “b” solution.
    • Adjust the pH 6.5 using KOH pellets.
    • Make up the final volume of the water to 200 ml and store it at 4-8 °C.
  • Add the following component in the given order in 950 ml of distilled water.
Components
Salt mix “lacking MgSO4 ” (20x stock)  50 ml
MgSO4 solution (0.4 M, 200x stock)  5 ml
Glucose  10 g
Trace element stock solution  1 ml
  • Mix it well in a 2 L flask. (Resulting pH will be a 6.6-No adjustment needed).

4.     Sordaria fimicola culturing methods

Various mediums are used to culture Sordaria in the labs such as Malt Extract Agar plus Copper (MEC), dichloran-rose bengal medium (DRBC), etc. The preparation of DRBC media is as follows:[1]

Dichloran-rose bengal medium (DRBC)

Procedure

  • Add the following components in the given order:
Components
The basal medium consisted of glucose 10 g
Peptone 5 g
MgSO4.7H20 0.5 g
K2HPO4 or KH2PO4 1.0 g
Agar 15 g
  • Sterilize the media.
  • Add sterilized chlortetracycline (10 µ/ml) to the media.
  • Add rose bengal (5% w/vol) to the solution.
  • Add Dichloran (2,6-dichloro-4-nitroaniline) solution (0.2%(w/v) in ethanol) to the solution.
  • Add agar to the solution and heat it to dissolve agar.
  • The pH of the solution will be at 5.6.

Methods to visualize fungal chromosomes

Modern cytogenetics techniques for chromosomal analysis include FISH, microarray FISH, multiplex-Karyotyping, etc. Here, we discuss the two most practiced techniques for the visualization of fungal chromosomes in labs. These techniques are: FISH (Fluorescent in situ hybridization) and Karyotyping.

Karyotyping by Germ Tube Burst Method  (GTBM)

For the karyotyping of fungal chromosomes, two methods are available: Pulsed-field gel electrophoresis (PFGE) and Germ Tube Burst Method  (GTBM). The application of PFGE involves genetic mapping, strain identification, analysis of transformed strains, and DNA preparation for genome analysis[2]. The Germ Tube Burst Method  (GTBM) is helpful in separating chromosomes of any size and in situ hybridization.

The Germ Tube Burst Method (GTBM) is as follows:[4]

Germ Tube Burst Method  (GTBM) (PFGE)

Materials

Poly- L-lysine solution (Dissolve 1 mg/mL Poly- L-Lysine in water), potato dextrose broth (PDB), rubber cement glue, Thiabendazole (50 mg/mL in dimethylsulfoxide (DMSO)) (1000x stock),  DAPI (4,6-diamidino-2-phenylindole) (1 mg/mL DAPI in antifade mounting solution)(1000x stock), antifade mounting solution, centrifuge, falcon tube, glass slides, miracloth, coverslip, and Epifluorescence microscope[4].

Sample Preparation
  1. Grow fungus in a solid culture medium for the production of conidia.
  2. Wash off the agar plates with sterile water to prepare a conidial suspension.
  3. Pass the conidial suspension through sterile Mira cloth to remove hyphal debris.
  4. Transfer the conidial suspension in a 50 ml falcon tube.
  5. Collect conidia for 5 minutes by centrifugation of suspension at 3,000–3,500 × g.
  6. Decant supernatant and wash the pellets twice by using sterilized water.
  7. Resuspend the conidia in PDB (potato dextrose broth) or any nutritive liquid media (concentration of conidia 2–3 × 10 5 conidia/mL).
  8. Put the slides in a poly- L-lysine solution (acts as an adhesive and reduce the DNA loss) and with gentle agitation, leave it in the solution 5-10 minutes.
  9. Make a rectangular frame on the slide by using rubber cement glue (this will prevent leaking of conidial suspension when it will be pipetted in the frame).
  10. Pipette 150–200 μL of conidial suspension inside the rectangular frame.
  11. Incubate the slide in a humid chamber (kept in dark-for conidial germination).
  12. Monitor the status of germination after 6 hours by using light microscopy.
  13. Pipette out the liquid from the slide surface (do not let it dry).
  14. Pipette out 200 μL of PDB containing thiabendazole (TBZ) solution (50 μg/mL) (mitosis arrest at metaphase).
  15. Add hydroxyurea (50mM)(increases metaphase frequency).
  16. Incubate the slide for 2–3 h at 22°C.
  17. Pipette out TBZ from the frame and remove rubber cement glue by using forceps.
  18. Dip the slide in sterile MQ to wash off TBZ.
  19. Wipe extra water using the filter paper.
  20.  Immerse the slide in fixation solution (methanol: glacial acetic acid 17:3, or 9:1 (v/v)) (prepare fresh) for 15–30 min.
  21. Dry the slide by passing over a smooth flame (do not overheat)[4].
Staining and observation
  1. Pipette 20–25 μL of 1 μg/mL DAPI solution onto the slide.
  2. Put a coverslip on the slide and keep it in darkness for 10-15 minutes.
  3. Observe chromosomes by using an epifluorescence microscope (with a 100× oil immersion objective lens)[4].

FISH method

To learn about the principle and application of Fluorescence in situ hybridization (FISH), read this article. Below, we discuss the steps to be followed for FISH of the filamentous fungi[6].

Materials

Hybridization Mixture, Biotin-14-dATP, 100 μg/ml RNase A,  SSC (saline sodium citrate buffer), ethanol, rubber cement, tween 20, DAPI, propidium iodide (PI), 50% (v/v) formamide, bovine serum albumin (BSA), 10 μg/ml avidin-fluorescein isothiocyanate (FITC), slide, and coverslip[6].

Hybridization Mixture[6]: A mix of 50% (v/v) deionized formamide, 10% (w/v) dextran sulfate in 2x SSC, 100 ng/μl sonicated salmon sperm DNA, and 2-5 ng/μl biotinylated probe DNA.

Procedure

Conidial and Cytological Preparation[6]

  1. Follow the Germ tube burst method (GTBM). (see Sample Preparation and Staining and observation sections)

DNA probe and labeling steps[6]

  1. Isolate plasmid DNAs by alkaline lysis method and purify it by using ethidium bromide-CsC1 density gradient centrifugation.
  2.  Label it with biotin-14-dATP by nick translation using the BioNick Labelling System.
  3. Mix the two labeled plasmid DNAs in equal amounts to use them as probes for FISH.

Fluorescence in situ hybridization steps[6]

  1. Prior to hybridization, treat the specimen with 100 μg/ml RNase A in 2x SSC (saline sodium citrate buffer) (lx SSC is 0.15 M NaC1, 0.015 M sodium citrate) for 1 h at 37 °C.
  2. Dehydrate it in ethanol series ((70%-85%-99%).
  3. Apply 15 μL of hybridization mixture on a slide and cover it with a coverslip.
  4. Seal the slide by using rubber cement.
  5. Denature the slide on a hot plate for 1.5 min at 78-80 °C.
  6. Incubate the slide for hybridization at 37°C for overnight (or 15 hr)
  7. After 15 hours, remove the rubber cement and coverslip in 2x SSC.
  8. Wash the slides twice in 50% (v/v) formamide (dissolved in 2x SSC) at 37 °C for 10 minutes each.
  9. Wash it twice again using 2x SSC at room temperature.
  10. Rinse the slide with 4x SSC (containing 0.05% (v/v) Tween 20).
  11. Block the slide using 3% (w/v) bovine serum albumin (BSA) in 4x SSC for 5 min at room temperature.
  12. Apply 40 μL of 10 μg/ml avidin-fluorescein isothiocyanate (FITC) (in 4xSSC) with 3% BSA.
  13. Put the coverslip on the slide and incubate it in a humid chamber at 37 °C for 1 hour.
  14. Wash the slides in  4x SSC with 0.05% Tween 20 at room temperature for 5 min each.
  15. Rinse the slides using 2x SSC.
  16. Mount the slides in fluorescence antifade solution containing 1 μg/ml DAPI and 1 μg/ml propidium iodide (PI).
  17. Observe the slides using epifluorescence microscopy[6].

Conclusion

Molecular cytogenetic techniques are a crucial tool to study the characteristic features of chromosomes and their functional roles in the organisms. Fungal cytogenetics gives us an insight into the diversity of the fungi living worldwide in all kinds of environments. They are a critical part of our ecosystem and play a big role in pharmaceuticals. There are many culture medium recipes available to culture fungi into labs. The selection of the media depends on the type of fungi to be analyzed whether it is yeast, mold, or filamentous fungi and type of experiments to be conducted. Karyotyping and FISH techniques are the current practices in labs for analyzing fungal chromosomes. While, technological advancement continues unraveling all the puzzles of fungi, their genetic diversity, and their clinical importance.

References

  1. A. Douglas King, Jr., Ailsa D. Hocking, and John I. Pitt (1979). Dichloran-Rose Bengal Medium for Enumeration and Isolation of Molds from Foods. Applied And Environmental Microbiology, 37(5), 959-964.
  2. Beadle, J., Wright, M., McNeely, L., & Bennett, J., (2003). Electrophoretic Karyotype Analysis in Fungi. Advances in Applied Microbiology, 243–270. DOI:10.1016/s0065-2164(03)53007-6
  3. Hill Terry and  Käfer E. (2001). Improved protocols for Aspergillus minimal medium: Trace element and minimal medium salt stock solutions. Fungal Genetics Newsletter, 48. DOI:10.4148/1941-4765.1173.
  4. Mehrabi, R., Taga, M., Aghaee, M., de Wit, P. J. G. M., & Kema, G. H. J. (2011). Karyotyping Methods for Fungi. Methods in Molecular Biology, 591–602. DOI:10.1007/978-1-61779-501-5_37
  5. Metzenberg, R. L. (2003). Vogel’s Medium N salts: avoiding the need for ammonium nitrate. Fungal Genetics Reports, 50 (6). DOI: https://doi.org/10.4148/1941-4765.1152.
  6. Masatoki Taga and Minoru Murata (1994). Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization. Chromosoma, 103, 408-413.
  7. Rodolfo Aramayo and Eric U. Selker (2013). Neurospora crassa, a Model System for Epigenetics Research. Cold Spring Harbor Laboratory Press. DOI: 10.1101/cshperspect.a017921
  8. Roshanak Salari, Rosita Salari (2017). Investigation of the Best Saccharomyces cerevisiae Growth Condition. Electronic Physician Journal, 1 (9), 3592-3597. DOI: http://dx.doi.org/10.19082/3592.
  9. Watkinson C. Sarah, Carlile J. Michael, and Gooday W. Graham (2001). The Fungi, 2nd edition, Academic Press, London.

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